HYBRIDIZATION AND IMMUNOHISTOCHEMISTRY METHOD
Silva-Filho, J.L.Q.1, Vasconcelos, J.L.A.1, Ferreira, A.S.1, Nascimento,J.C.F.1,
Cavalcanti, C.L.B.1, Beltrão, E.I.C.1,2
1 Grupo de Pesquisa Biomarcadores no Câncer (BmC) - Universidade Federal de Pernambuco, Recife, PE, Brasil.
2 Departamento de Bioquímica – Centro de Biociências (CBC) - Universidade Federal de Pernambuco, Recife, PE, Brasil.
Correspondence: Eduardo Beltrão. LIKA – UFPE. Av. Prof. Moraes Rêgo, s/n – CDU, Recife, PE – Brasil. 50670-901. E-mail: [email protected]
ABSTRACT
Background: Breast cancer is the second most common cancer worldwide among
women, accounting for 25% of all female cancers. In Brazil, it is the most incident cancer in women. The study of some markers may help the understanding of disease progression, as well as planning the most appropriate therapy. C-myc gene is involved in cell differentiation, apoptosis, angiogenesis and immortalization, playing central role in the tumorgenic process and its amplification is associated with invasive ductal carcinomas (IDC). This study aimed to evaluate c-myc amplification and its expression prein using chromogenic in situ hybridization (CISH) and immunohistochemistry to correlate theses molecular markers with clinicopathological features. Materials and
Methods: A retrospective analysis of breast cancer subtypes, age, tumor status nodal,
staging pathological, histopathological and nuclear grading of 60 cases. The estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor- 2 (HER2), and Ki-67 of tumor samples were all investigated by immunohistochemistry according to the methods used to classify breast cancer subtypes as proposed in the
St. Gallen Consensus Report, 2013. To evaluate the c-myc copy number and its
protein expression, CISH and IHC analyses were performed for breast cancer, in Brazilian. Results: The most frequent molecular sub-types of IDC were triple-negative (40%) and Luminal A (37.7%). Luminal A (86,4%) and B (83,3%) sub-types showed to be well differentiated tumours regarding histological grade (p=0.0135) and tumors size (≤5,0cm) at diagnostic ((p=0,0159). On the contrary, the types superexpressed HER-2 and triplo nevative carcinomas presented larger (p=0.0159) and poorly differentiated tumors (p= 0.0570) with linfonodes involvement and advanced stages at the time of diagnosis. We observed c-myc oncogene amplification in 21 tumours (35%) and positive protein expression in 46 tumors (76,7%). No statistical difference between amplified and non-amplified and positive and negative molecular subtypes of IDC was found. Conclusion: A better understanding of tumors behavior of
c-myc amplification and protein expression and its clinical impact, combined with
other molecular parameters, will help to stratify patients into risk groups and to conduct more targeted therapies.
Key Words: Breast cancer, c-myc, gene amplification, expression, chromogenic in situ hybridization, immunohistochemistry.
INTRODUCTION
Breast cancer is the second most frequent type of cancer in the world. It is the most common cancer in feminine gender, corresponding to 25% of women cancer cases.1,2 In Brazil, it was estimated that 600 mil cancer cases will be
detected in 2016, of which 57.690 will be breast cancer.3
Breast cancer etiology is unknown; nevertheless, some risk factors such as precocious menarche, late menopause, nulliparity, women age in their first gestation, contraceptive use, hormonal reposition therapy, exposition to ionizing radiation and genetic predisposition could be involved with the disease development.4
Usually, invasive ductal carcinoma has an aggressive clinic behavior and variations in therapeutic responses depending of staging as well as presence or absence of the expression certain biomarkers.4,5,6
Numerical and structural alterations in chromosome 8 have been commonly detected in breast cancer7,8,9 and in others tumors types.10,11,12 The majority of
these modifications occur as low level changes in copies numbers, including complete or partial deletions in 8p and gains in 8q chromosome arms.13,14 High
amplification levels have been found not only in 8q24 but in other regions of 8 chromosome, as 8p12 and 8q21.15,16,17
Gene amplification is an important mechanism for protein overexpression as well as for oncogenes activation in cancer.18 C-myc proto-oncogene (homolog
to v-myc viral oncogene which causes bird myelocytomatosis), situated in 8 chromosome long arm19, is generally an amplification target.20 The amplification
can be visualized as double minutes (DM) and/or homogeneous staining region (HSR).21 C-myc gene product is a transcription factor involved in cell proliferation,
division, growth, metabolism, differentiation and apoptosis. Deregulation of c-myc gene can induces carcinogenesis by genomic instability, cell proliferation control, escape from immune system surveillance and immortalization.22,23
Amplification and/or overexpression of c-myc oncogene has been detected in different neoplasies, including breast cancer.24-31 In breast tumors, c-myc
amplification frequency, mRNA and protein overexpression vary from 1 to 94%, 22 to 95% and 50 to 100% respectively.32,33 In spite of these data, c-myc amplification
frequency is not so evident, which variation depends on the type of the studies and the employed technique.34
Unlike HER-2 oncogene amplification and overexpression, which already exists a consensus with regard to it clinic application and it therapeutic impact.35,36,37, c-myc amplification pattern is not well established to diagnostic and
therapeutic uses. Some studies demonstrated that c-myc amplification is associated with more aggressive phenotypes38 and poor prognostic in the
presence of molecular marker.39,26,29
Nowadays, some groups are studying the use of c-myc in association with others biomarkers for evaluate therapeutic response29 as well as tumors
stratification in different risk groups26, which, illustrates the relevant role of c-myc
gene in breast cancer. This study aimed to evaluate c-myc amplification and its expression prein using chromogenic in situ hybridization (CISH) and immunohistochemistry to correlate theses molecular markers with clinicopathological features.